KRAS: ONE ACTOR, MANY POTENTIAL ROLES IN DIAGNOSIS

Loredana Bruno

UNIVERSITÀ DEGLI STUDI DI PALERMO

Scuola di Specializzazione in Biochimica Clinica

Direttore Prof. Marcello Ciaccio

Proto-oncogene on chromosome 12 (12p12)

Encodes a 188aa protein with catalytic activity

Protein is a 21 KDa GTP-protein and play a role in cell

proliferation, cell differentiation, apoptosis

Activating mutations in codons 12, 13, 59, 61, 117, 146

KRAS gene

Mutations hot-spots

59 61

5’ 3’

ex2 ex3 ex4 ex5 ex6 ex1

12

13 117 146

• Ras family genes (K-H-N Ras) are the most common targets for somatic gain-of-function mutations in human cancers

• Activating RAS mutations occur in 30% of human cancers – Specific RAS genes are mutated in different

cancers

• KRAS is most frequently mutated in cancer (pancreatic, colorectal, lung, endometrial, and cervical cancers)

KRAS and cancer

EGFR

NUCLEUS

LIGAND

PIP3 MEK ERK

PTEN

RAS

B-RAF

PI3K

PIP2

MAPK AKT

mTOR

e.g. EGF, TGFa, Amphiregulin, Epiregulin

METASTASIS

PROLIFERATION SURVIVAL

ANGIOGENESIS

e.g. VEGF

EGFR pathway map

KRAS: a biomarker of……...

Prognosis

Therapy

Diagnosis

Molecular Diagnostic in laboratory medicine

KRAS: Prognostic Role

• RASCAL study multivariate analysis presence of KRAS mutation associated with increased risk of recurrence and death (Andreyev et al. 1998)

– Only Gly12Val – RASCAL II confirmed but only in Dukes C (Stage

3) tumors (Andreyev et al. 2001) • Others studies have not confirmed

E.R. Fearon, B. Vogelstein. Cell 1990

NormalEpithelium Dysp lastic ACF Ear lyAdenoma LateAdenoma Carcinoma MetastasisIntermediate Adenoma

APC/ -catenin K-RAS 18q p53 OtherChanges?

NormalEpithelium

Dysplastic

ACF EarlyAdenoma

Late

AdenomaCarcinoma Metastasis

Intermediate Adenoma

APC/ -catenin K-RAS 18q p53Other

Changes?

PIK3CA BRAF

Genetic Model for Colorectal Tumorigenesis

(35-45%)

KRAS: Predictive Role in therapy

KRAS mutations have emerged as a major predictor of resistance to panitumumab or cetuximab in patients with metastatic colorectal cancer

Amado et al. J Clin Oncol 2008;

Bokemeyer C et al. 2009 and 2011

Van Custem E et al. 2009 and 2011

AIOM society has recommended determination of KRAS mutation status in all patients with metastatic colorectal cancer who are candidates for anti-EGFR therapy (www.aiom.it)

Genetic testing includes research of mutation in exons 2-3-4 of KRAS and NRAS genes

SURVIVAL

ANGIOGENESIS

e.g. VEGF

PI3K

PIP2

PIP3 PTEN

AKT

mTOR

NUCLEUS

LIGAND

EGFR

Blocks EGFR dimerization

METASTASIS

PROLIFERATION

RAF

MEK ERK

MAPK

RAS

Wt

Anti EGFR Abs (Cetuximab, Panitumumab)

Anti-EGFR Abs: KRAS Wild Type

SURVIVAL

ANGIOGENESIS

e.g. VEGF

PI3K

PIP2

PIP3 PTEN

AKT

mTOR

NUCLEUS

LIGAND

EGFR

Blocks EGFR dimerization

METASTASIS

PROLIFERATION

MEK ERK

MAPK

RAF

RAS

Mutation

Anti EGFR Abs (Cetuximab, Panitunumab)

Anti-EGFR Abs: KRAS mutation

KRAS as diagnostic tool for pancreatic cyst lesions:

our experience

• Pancreatic cyst lesions may be incidentally detected by abdominal imaging in up to 13,5% of patients for reason unrelated to the pancreas (Lee et al. 2010)

• Simple (retention) cysts or pseudocysts or serous cystadenomas lack malignant potential

• Mucinous cystic neoplasms (MCN) and intraductal papillary mucinous neoplasm (IPMN) have a malignant potential and may require surgical treatment

• Various combinations of morphology, cytology and tumor markers could not provide diagnostic accuracy (Brugge WR et al 2004)

• CEA levels (>192ng/mL) can discriminate between mucinous vs non-mucinous (79% accuracy) (Brugge WR et al 2004)

• KRAS mutations alone had a sensitivity of 45% and specificity of 96% for diagnosing mucinous cysts (Khalid A et al. 2009)

KRAS: Diagnostic role

DNA extraction

Pancreatic Cyst fluid

EUS-FNA

Methods

Biochemical markers dosage

(CEA, CA19.9 etc)

Direct Automated Sequencing (KRAS exons 2 and 3)

Results: part 1

G12D

G12R

G12V

G13D

Q61H

Q61K

Q61L

Mutated

Wild Type

41% 59%

Type of Mutation N %

G12D 8 40

G12R 3 15

G12V 5 25

G13D 1 5

Q61H 1 5

Q61K 1 5

Q61L 1 5

n.50 (2011-2014)

Results: part 2

KRAS ex 2 KRAS ex 3 Diagnosi istologica

1 G12D Tumore Maligno Testa del Pancreas

2 G12D IPMN

3 G12D lesione cistica Non Valutabile

4 G12D IPMN

5 G12D Tumore Maligno testa del pancreas

6 G12D IPMN

7 G12D IPMN

8 G12D IPMN

9 G12R IPMN

10 G12R IPMN

11 G12R IPMN

12 G12V Tumore Maligno testa del pancreas

13 G12V IPMN

14 G13D IPMN

15 n Q61H IPMN

16 n Q61K pseudocisti

17 n Q61L IPMN

18 G12V Tumore Maligno testa del pancreas

19 G12V IPMN

20 G12V IPMN

Results: part 3

KRAS Wild type (n. 30) KRAS mutant (n.20)

Sex - Female - Male

13 17

12 8

CEA (>192 ng/mL) n.d.

12 6

8 3

Mucinous Non-mucinous

12

18

18 2

Codon 12 Mutant Codon 13 Mutant Codon 61 Mutant

- - -

16 1 3

GNAS codon 201 Mutant (performed in n.30 patient)

2 5

90% specificity

Conclusions

The current diagnosis of pancreatic cyst type does not reliably allow to distinguish between non-mucinous vs mucinous cysts and non malignant vs potential malignant cysts

KRAS is a useful diagnostic tool for identification of pancreatic cyst with mucinous histotype

KRAS mutations often are more sensitive than CEA levels

New high sensibility methods are needed for KRAS mutational analysis

Or………new biomarkers?